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  • 99
    Thermo Fisher sirnas
    SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated <t>siRNAs</t> targeting NPC1 , TMEM97 or <t>NPC2</t> . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P
    Sirnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirnas/product/Thermo Fisher
    Average 99 stars, based on 9799 article reviews
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    sirnas - by Bioz Stars, 2019-05
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    99
    Thermo Fisher sirna
    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting <t>siRNA</t> continued. Deconvolution microscopic images (single optical sections) of <t>HeLa</t> cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna/product/Thermo Fisher
    Average 99 stars, based on 16005 article reviews
    Price from $9.99 to $1999.99
    sirna - by Bioz Stars, 2019-05
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    91
    Santa Cruz Biotechnology p65 sirna
    The effect of MG132 protease inhibitor and <t>p65</t> <t>siRNA</t> on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p
    P65 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p65 sirna/product/Santa Cruz Biotechnology
    Average 91 stars, based on 58 article reviews
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    p65 sirna - by Bioz Stars, 2019-05
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    87
    OriGene shrna sequence targeting mt1 mmp
    <t>MT1-MMP</t> overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm
    Shrna Sequence Targeting Mt1 Mmp, supplied by OriGene, used in various techniques. Bioz Stars score: 87/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    GenePharma Company anti survivin sirna
    Synthesis of NDCONH(CH 2 ) 2 <t>NH-VDGR/survivin-siRNA.</t> Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.
    Anti Survivin Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti survivin sirna/product/GenePharma Company
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    80
    GenePharma Company fluorescein labeled vegf sirna
    <t>VEGF</t> protein expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; <t>siRNA,</t> small interfering RNA; VEGF, vascular endothelial growth factor.
    Fluorescein Labeled Vegf Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labeled vegf sirna/product/GenePharma Company
    Average 80 stars, based on 5 article reviews
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    78
    GenePharma Company fluorescein labeled survivin sirna
    Synthesis of NDCONH(CH 2 ) 2 <t>NH-VDGR/survivin-siRNA.</t> Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.
    Fluorescein Labeled Survivin Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labeled survivin sirna/product/GenePharma Company
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    77
    Thermo Fisher tet1 sirna
    Nanog overexpression or suppression of MAPK/ERK signaling rescues <t>Tet1</t> KD phenotype. ( A ) Western blot analysis showing Nanog overexpression with HA-tag. ( B ) AP staining of E14Tg2a mESCs, with and without Nanog overexpression, transfected with control <t>siRNA</t> and Tet1 siRNA #1. Cells were cultured in normal ESC medium, and AP staining was performed 96 h after transfection. ( C ) Relative mRNA levels of selected mESC pluripotency genes and differentiation marker genes in control and Tet1 KD mESCs in 2i medium. Oct4GiP cells were transfected with control siRNA or Tet1 siRNA #1 at 50 nM in 24-well plates in 2i-medium (which inhibits MAPK/ERK and Gsk-3b signaling) and cells were harvested 96 h after transfection. The mRNA levels in control cells are set as one. Data are normalized to Actin . Error bars represent SEM of three experiments.
    Tet1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated siRNAs targeting NPC1 , TMEM97 or NPC2 . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P

    Journal: Human Molecular Genetics

    Article Title: Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

    doi: 10.1093/hmg/ddw204

    Figure Lengend Snippet: SiRNA-mediated knockdown of TMEM97 increases NPC1 protein levels, ameliorates cholesterol accumulation and restores cholesterol delivery to the ER in NPC1 -deficient HeLa cells. ( A ) Cultured HeLa cells were transfected for 48h with either control siRNA or indicated siRNAs targeting NPC1 , TMEM97 or NPC2 . Whole cell lysates were subjected to Western blotting and probed with antibodies against NPC1 and β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels of control siRNA treated cells ( n = 3 independent experiments per condition; ** P

    Article Snippet: For RNA interference the following siRNAs were used: For knockdown of TMEM97 : siRNAs #140160 (I) (Ambion) and #SI03198314 (II) (QIAGEN); for knockdown of NPC1 : siRNAs #114041 (I) and #106017 (II) (both from Ambion); for knockdown of NPC2 : siRNAs #135801 (I) and #135802 (II) (both from Ambion); as a non-silencing negative control: scrambled-siRNA #4611 (Ambion).

    Techniques: Cell Culture, Transfection, Western Blot

    Reduction of TMEM97 elevates residual mutant NPC1 protein levels and restores cholesterol trafficking in NPC1 -mutant fibroblasts. (A) Primary human skin fibroblasts homozygous or compound-heterozygous for indicated NPC1 alleles (in brackets, see Supplementary Material, Table S1 for details) were transfected with NPC1 or TMEM97 siRNAs. Ninety-six hours after transfection, whole cell lysates were subjected to Western blotting and analyzed for NPC1 or β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels in control siRNA treated cells ( n = 2 independent experiments; mean±SEM, * P

    Journal: Human Molecular Genetics

    Article Title: Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

    doi: 10.1093/hmg/ddw204

    Figure Lengend Snippet: Reduction of TMEM97 elevates residual mutant NPC1 protein levels and restores cholesterol trafficking in NPC1 -mutant fibroblasts. (A) Primary human skin fibroblasts homozygous or compound-heterozygous for indicated NPC1 alleles (in brackets, see Supplementary Material, Table S1 for details) were transfected with NPC1 or TMEM97 siRNAs. Ninety-six hours after transfection, whole cell lysates were subjected to Western blotting and analyzed for NPC1 or β-actin. NPC1 protein levels were quantified as a ratio to β-actin and normalized to levels in control siRNA treated cells ( n = 2 independent experiments; mean±SEM, * P

    Article Snippet: For RNA interference the following siRNAs were used: For knockdown of TMEM97 : siRNAs #140160 (I) (Ambion) and #SI03198314 (II) (QIAGEN); for knockdown of NPC1 : siRNAs #114041 (I) and #106017 (II) (both from Ambion); for knockdown of NPC2 : siRNAs #135801 (I) and #135802 (II) (both from Ambion); as a non-silencing negative control: scrambled-siRNA #4611 (Ambion).

    Techniques: Mutagenesis, Transfection, Western Blot

    Knockdown of TMEM97 increases NPC1 protein levels in lysosomal and extra-lysosomal compartments. ( A ) Confocal images from HeLa cells treated with indicated siRNAs for 48h and stained for NPC1 with an NPC1-specific antibody ( 32 ) (scale bar = 10µm). Box plots show medians (lines), lower and upper quartiles (boxes), 10th and 90th percentiles (whiskers) and outliers (circles) of NPC1 signal intensities per cell as quantified automatically from stacks of confocal images using CellProfiler software ( n = 5 independent experiments; *** P

    Journal: Human Molecular Genetics

    Article Title: Reduction of TMEM97 increases NPC1 protein levels and restores cholesterol trafficking in Niemann-pick type C1 disease cells

    doi: 10.1093/hmg/ddw204

    Figure Lengend Snippet: Knockdown of TMEM97 increases NPC1 protein levels in lysosomal and extra-lysosomal compartments. ( A ) Confocal images from HeLa cells treated with indicated siRNAs for 48h and stained for NPC1 with an NPC1-specific antibody ( 32 ) (scale bar = 10µm). Box plots show medians (lines), lower and upper quartiles (boxes), 10th and 90th percentiles (whiskers) and outliers (circles) of NPC1 signal intensities per cell as quantified automatically from stacks of confocal images using CellProfiler software ( n = 5 independent experiments; *** P

    Article Snippet: For RNA interference the following siRNAs were used: For knockdown of TMEM97 : siRNAs #140160 (I) (Ambion) and #SI03198314 (II) (QIAGEN); for knockdown of NPC1 : siRNAs #114041 (I) and #106017 (II) (both from Ambion); for knockdown of NPC2 : siRNAs #135801 (I) and #135802 (II) (both from Ambion); as a non-silencing negative control: scrambled-siRNA #4611 (Ambion).

    Techniques: Staining, Software

    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity. Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity. Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA. ( A–B ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). ( C–D ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA. ( A–B ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). ( C–D ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Knockdown of Nups and NTRs in HOS cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in HOS cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Transfection, Generated, Infection

    Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity. Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity. Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Knockdown of Nups and NTRs in HT1080 cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. (n/a – not tested since HT1080 cells do not express NUP210, see Figure 2—figure supplement 1 ). Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in HT1080 cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. (n/a – not tested since HT1080 cells do not express NUP210, see Figure 2—figure supplement 1 ). Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Knockdown of Nups and NTRs in non-dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot is generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown in . The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in non-dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot is generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown in . The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye DRAQ5. For each plot, 3000–12000 events were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye DRAQ5. For each plot, 3000–12000 events were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection. ( A ) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. ( B ) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. ( D ) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection. ( A ) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. ( B ) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. ( D ) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Expressing, Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Effect of Nup and NTR depletion on HIV-1 infection in HOS cells. ( A ) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 infection in HOS cells. ( A ) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Transfection

    Knockdown of Nups and NTRs in dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 6 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 6 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing

    Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye propidium iodide (PI). For each plot, 3000–12000 events per sample were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye propidium iodide (PI). For each plot, 3000–12000 events per sample were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 5 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 5 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing

    Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effects of depleting individual Nups on NPC integrity and function, and MX2 localization. Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6 , and Figure 6—figure supplements 1 – 18 . Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effects of depleting individual Nups on NPC integrity and function, and MX2 localization. Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6 , and Figure 6—figure supplements 1 – 18 . Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA. ( A–C ) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of 5 μM CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). X-axis legend for A-B is below graph B, x-axis legend for C-D is below graph D. ( D ) Infectivity of HIV-1 N57S GFP reporter virus in HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA. ( A–C ) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of 5 μM CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). X-axis legend for A-B is below graph B, x-axis legend for C-D is below graph D. ( D ) Infectivity of HIV-1 N57S GFP reporter virus in HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity. Infectivity of ( A ) EIAV or ( B-D ) FIV GFP reporter virus in ( A-B ) HeLa or ( C ) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or ( D ) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity. Infectivity of ( A ) EIAV or ( B-D ) FIV GFP reporter virus in ( A-B ) HeLa or ( C ) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or ( D ) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR

    The effect of MG132 protease inhibitor and p65 siRNA on Nestin gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Nestin mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Nestin gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Nestin mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR

    The effect of MG132 protease inhibitor and p65 siRNA on Dentin Sialophosphoprotein (DSPP) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA (A and E, respectively), cytokine dose alone (B), and cytokine dose in the presence of MG132 or p65 siRNA (C and F, respectively) for 7 days for the examination of odontoblastic gene marker DSPP. The odontoblastic cell morphology was examined using scanning electron microscopy after 12 days (D) (for treatment IL-1β (0.5) + TNFα (1) + MG132 (0.5)). DSPP mRNA was assessed using RT-PCR. Data are presented as the mean + S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Dentin Sialophosphoprotein (DSPP) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA (A and E, respectively), cytokine dose alone (B), and cytokine dose in the presence of MG132 or p65 siRNA (C and F, respectively) for 7 days for the examination of odontoblastic gene marker DSPP. The odontoblastic cell morphology was examined using scanning electron microscopy after 12 days (D) (for treatment IL-1β (0.5) + TNFα (1) + MG132 (0.5)). DSPP mRNA was assessed using RT-PCR. Data are presented as the mean + S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Marker, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction

    The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression with low- and high-cytokine doses. Cells were exposed to various MG132 or p65 siRNA concentrations for 7 days. NF-κB mRNA (A, B) was assessed using qRT-PCR. NF-κB p65 protein (C, D) was assessed using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression with low- and high-cytokine doses. Cells were exposed to various MG132 or p65 siRNA concentrations for 7 days. NF-κB mRNA (A, B) was assessed using qRT-PCR. NF-κB p65 protein (C, D) was assessed using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    The effect of MG132 protease inhibitor and p65 siRNA on Alkaline Phosphatase (ALP) protein expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. ALP protein was assayed using SensoLyte ALP Assay. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Alkaline Phosphatase (ALP) protein expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. ALP protein was assayed using SensoLyte ALP Assay. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, ALP Assay, Expressing

    The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression. Cells were exposed to various MG132 or p65 siRNA doses for 7 days. NF- κB mRNA (A, B) was assayed using qRT-PCR. NF-κB p65 protein expression (C, D) was evaluated using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicates statistical comparison with control result. Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by * for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression. Cells were exposed to various MG132 or p65 siRNA doses for 7 days. NF- κB mRNA (A, B) was assayed using qRT-PCR. NF-κB p65 protein expression (C, D) was evaluated using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicates statistical comparison with control result. Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by * for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    The effect of MG132 protease inhibitor and p65 siRNA on collagen matrix formation in DPSCs with low- and high-cytokine doses. Bioquant analysis of collagen formation DPSC after 9 days’ treatment. DPSCs exposed to various inflammatory treatments in the presence or absence of MG132 (A and B) or p65 siRNA (C and D) were analyzed from picrosirius staining images using the Bioquant software. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on collagen matrix formation in DPSCs with low- and high-cytokine doses. Bioquant analysis of collagen formation DPSC after 9 days’ treatment. DPSCs exposed to various inflammatory treatments in the presence or absence of MG132 (A and B) or p65 siRNA (C and D) were analyzed from picrosirius staining images using the Bioquant software. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Staining, Software

    MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Multiple Displacement Amplification, Immunofluorescence, Confocal Microscopy

    MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Immunofluorescence, Confocal Microscopy, Stable Transfection, Expressing

    Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Incubation

    Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Multiple Displacement Amplification, Migration, Expressing, Transwell Migration Assay, Incubation

    Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Migration, In Vivo, Activation Assay, Incubation

    MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Imaging, Migration

    Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: In Situ, Transformation Assay

    MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Chick Chorioallantoic Membrane Assay, Multiple Displacement Amplification, Stable Transfection, Expressing, Fluorescence

    MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Zymography, Incubation, Activation Assay, Transwell Migration Assay

    Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Migration, Real-time Polymerase Chain Reaction, Zymography, Activation Assay, Transfection, Incubation, Recombinant

    MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Zymography, Incubation

    MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, In Vivo, Stable Transfection, Injection, Chick Chorioallantoic Membrane Assay, Labeling, Isolation, Confocal Microscopy

    Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Migration, Activation Assay, Incubation, Real-time Polymerase Chain Reaction, Derivative Assay, Stable Transfection, shRNA, Construct, Expressing, Transwell Migration Assay

    MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Multiple Displacement Amplification, Incubation, Immunofluorescence

    Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Microscopy, Small Interfering RNA

    The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Electron Microscopy, Small Interfering RNA

    Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation

    Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Mass Spectrometry, Small Interfering RNA

    TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Transmission Electron Microscopy, Transmission Assay, Electron Microscopy, Small Interfering RNA

    Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation, Enzyme-linked Immunosorbent Assay

    Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    VEGF protein expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: VEGF protein expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Expressing, Small Interfering RNA

    Scheme for GPF and GPF/DOX/VEGF-siRNA preparation. (I) KOH, 70°C, 24 hours, DI water. (II) Room temperature, 24 hours. (III) Stirred at room temperature, without light, 12 hours, DI water. (IV) Incubation at 37°C, 30 minutes, DEPC water. Abbreviations: DEPC, diethyl pyrocarbonate; DI, deionized; DOX, doxorubicin; FA, folic acid; GO, graphene oxide; GPF, GO-PLL/FA; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Scheme for GPF and GPF/DOX/VEGF-siRNA preparation. (I) KOH, 70°C, 24 hours, DI water. (II) Room temperature, 24 hours. (III) Stirred at room temperature, without light, 12 hours, DI water. (IV) Incubation at 37°C, 30 minutes, DEPC water. Abbreviations: DEPC, diethyl pyrocarbonate; DI, deionized; DOX, doxorubicin; FA, folic acid; GO, graphene oxide; GPF, GO-PLL/FA; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Incubation, Small Interfering RNA

    Confocal images of the HeLa cells. ( A ) Blank control, ( B ) naked FAM-VEGF-siRNA, ( C ) DOX, ( D ) GPF/FAM-VEGF-siRNA, ( E ) GPF/DOX, ( F ) Lipo™2000/FAM-VEGF-siRNA, and ( G ) GPF/DOX/FAM-VEGF-siRNA. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Confocal images of the HeLa cells. ( A ) Blank control, ( B ) naked FAM-VEGF-siRNA, ( C ) DOX, ( D ) GPF/FAM-VEGF-siRNA, ( E ) GPF/DOX, ( F ) Lipo™2000/FAM-VEGF-siRNA, and ( G ) GPF/DOX/FAM-VEGF-siRNA. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Image of tumors of blank control, positive control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Image of tumors of blank control, positive control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Positive Control, Small Interfering RNA

    The tumor weights of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, DOX (positive control), and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: The tumor weights of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, DOX (positive control), and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Positive Control, Small Interfering RNA

    The tumor volume of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: The tumor volume of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Degradation of GPF/VEGF-siRNA with heparin and anti-RNase A. Abbreviations: GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Degradation of GPF/VEGF-siRNA with heparin and anti-RNase A. Abbreviations: GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Agarose gel electrophoresis retardation assays of VEGF-siRNA complexed with GPF ( A ) and GO ( B ). Abbreviations: GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Agarose gel electrophoresis retardation assays of VEGF-siRNA complexed with GPF ( A ) and GO ( B ). Abbreviations: GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    VEGF mRNA expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: VEGF mRNA expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Expressing, Small Interfering RNA

    Zeta potential measurements of GO ( A ), GO-PLL ( B ), GPF ( C ), and GPF/DOX/VEGF-siRNA ( D ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-PLL/folic acid; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Zeta potential measurements of GO ( A ), GO-PLL ( B ), GPF ( C ), and GPF/DOX/VEGF-siRNA ( D ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-PLL/folic acid; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Anti-proliferation effect of GPF/DOX/VEGF-siRNA on HeLa cells (n=3). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Anti-proliferation effect of GPF/DOX/VEGF-siRNA on HeLa cells (n=3). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    The expression of VEGF protein in vivo (n=5). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: The expression of VEGF protein in vivo (n=5). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Expressing, In Vivo, Small Interfering RNA

    TEM images of GO ( A ), GPF ( B ), and GPF/DOX/VEGF-siRNA ( C ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: TEM images of GO ( A ), GPF ( B ), and GPF/DOX/VEGF-siRNA ( C ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Transmission Electron Microscopy, Small Interfering RNA, Transmission Assay, Electron Microscopy

    Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Microscopy, Small Interfering RNA

    The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Electron Microscopy, Small Interfering RNA

    Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation

    Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Mass Spectrometry, Small Interfering RNA

    TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Transmission Electron Microscopy, Transmission Assay, Electron Microscopy, Small Interfering RNA

    Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation, Enzyme-linked Immunosorbent Assay

    Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Nanog overexpression or suppression of MAPK/ERK signaling rescues Tet1 KD phenotype. ( A ) Western blot analysis showing Nanog overexpression with HA-tag. ( B ) AP staining of E14Tg2a mESCs, with and without Nanog overexpression, transfected with control siRNA and Tet1 siRNA #1. Cells were cultured in normal ESC medium, and AP staining was performed 96 h after transfection. ( C ) Relative mRNA levels of selected mESC pluripotency genes and differentiation marker genes in control and Tet1 KD mESCs in 2i medium. Oct4GiP cells were transfected with control siRNA or Tet1 siRNA #1 at 50 nM in 24-well plates in 2i-medium (which inhibits MAPK/ERK and Gsk-3b signaling) and cells were harvested 96 h after transfection. The mRNA levels in control cells are set as one. Data are normalized to Actin . Error bars represent SEM of three experiments.

    Journal: Nucleic Acids Research

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

    doi: 10.1093/nar/gkr1253

    Figure Lengend Snippet: Nanog overexpression or suppression of MAPK/ERK signaling rescues Tet1 KD phenotype. ( A ) Western blot analysis showing Nanog overexpression with HA-tag. ( B ) AP staining of E14Tg2a mESCs, with and without Nanog overexpression, transfected with control siRNA and Tet1 siRNA #1. Cells were cultured in normal ESC medium, and AP staining was performed 96 h after transfection. ( C ) Relative mRNA levels of selected mESC pluripotency genes and differentiation marker genes in control and Tet1 KD mESCs in 2i medium. Oct4GiP cells were transfected with control siRNA or Tet1 siRNA #1 at 50 nM in 24-well plates in 2i-medium (which inhibits MAPK/ERK and Gsk-3b signaling) and cells were harvested 96 h after transfection. The mRNA levels in control cells are set as one. Data are normalized to Actin . Error bars represent SEM of three experiments.

    Article Snippet: RNAi experiments were performed using indicated individual siRNAs: Tet1 siRNA #1 (Invitrogen, MSS284895), Tet1 siRNA #2 (Invitrogen, MSS284897), Tet1 siRNA #3 (Dharmacon, D-062861-01), Tet1 siRNA #4 (Dharmacon, D-062861-02), Tet1 siRNA #5 (Dharmacon, D-062861-03), Tet1 siRNA #6 (Dharmacon, D-062861-04) and control siRNA duplex targeting firefly luciferase (Dharmacon, 5′-CGTACGCGGAATACTTCGA).

    Techniques: Over Expression, Western Blot, Hemagglutination Assay, Staining, Transfection, Cell Culture, Marker